Airborne Exposure of the Cornea to PM10 Induces Oxidative Stress and Disrupts Nrf2 Mediated Anti-Oxidant Defenses

The purpose of this study is to test the effects of whole-body animal exposure to airborne particulate matter (PM) with an aerodynamic diameter of <10 μm (PM10) in the mouse cornea and in vitro. C57BL/6 mice were exposed to control or 500 µg/m3 PM10 for 2 weeks. In vivo, reduced glutathione (GSH) and malondialdehyde (MDA) were analyzed. RT-PCR and ELISA evaluated levels of nuclear factor erythroid 2-related factor 2 (Nrf2) signaling and inflammatory markers. SKQ1, a novel mitochondrial antioxidant, was applied topically and GSH, MDA and Nrf2 levels were tested. In vitro, cells were treated with PM10 ± SKQ1 and cell viability, MDA, mitochondrial ROS, ATP and Nrf2 protein were tested. In vivo, PM10 vs. control exposure significantly reduced GSH, corneal thickness and increased MDA levels. PM10-exposed corneas showed significantly higher mRNA levels for downstream targets, pro-inflammatory molecules and reduced Nrf2 protein. In PM10-exposed corneas, SKQ1 restored GSH and Nrf2 levels and lowered MDA. In vitro, PM10 reduced cell viability, Nrf2 protein, and ATP, and increased MDA, and mitochondrial ROS; while SKQ1 reversed these effects. Whole-body PM10 exposure triggers oxidative stress, disrupting the Nrf2 pathway. SKQ1 reverses these deleterious effects in vivo and in vitro, suggesting applicability to humans.


Introduction
Air pollution is a major contributor to health problems worldwide [1]. The World Health Organization (WHO) air quality model demonstrates that ambient air pollution annually causes 4.2 million deaths, and 91% of the world's population lives in places where air quality exceeds the limits of WHO guidelines [2]. Epidemiological evidence shows adverse health effects associated with exposure to airborne particulate matter with a mean aerodynamic diameter of <10 µm (PM 10 ) [3]. Long-term exposure is associated with increased risk of cardiovascular [4], cerebrovascular [5], pulmonary diseases [6], arteriosclerosis [7], and cancer [8], while short-term exposure can lead to asthma [9], bronchitis [10], and other respiratory ailments [11]. The effects of PM 10 have largely focused on the lungs [12][13][14]. However, very little is known about the effects of PM 10 on the cornea, which is also readily exposed to this pollutant. Epidemiological and clinical data suggest that air pollution in which PM is a major constituent can cause transient ocular allergies (redness, discharge, foreign body sensation, and itching) [15]. Clinically, dry eye caused by particulates, is a global problem [16]. In this regard, studies from South Korea showed that high PM 10 exposure correlated with increased outpatient visits for ocular diseases, including emergency room visits for keratitis [17]. A recent study in mice examined the effects of air pollution in Argentina and found that exposure to polluted air compromised corneal immunity and worsened inflammation in acute herpes simplex keratitis [18]. In fact, dry eye [19][20][21][22] and conjunctivitis [23,24] are linked (causative or makes the disease

PM 10 Exposure: Tear Secretion and Corneal Sensitivity
The effects of whole-body exposure to PM 10 vs. control on tear secretion and corneal sensitivity at 0 (pre-exposure) and 2 weeks (post-exposure) are shown in Figure 1A-C. No differences in tear volume of PM 10 vs. control exposed mice were detected at the 2 weeks time period as observed by the phenol-red thread images ( Figure 1A) and tear volume represented as a bar graph in Figure 1B. No significant differences in corneal sensitivity were observed between PM 10 vs. control exposed groups ( Figure 1C) at 2 weeks of exposure.

Effects of PM 10 on GSH and MDA Levels and Histopathology
Levels of GSH were significantly lower (p < 0.05) after 2 weeks of exposure to PM 10 vs. control (Figure 2A). Levels of MDA ( Figure 2B) were significantly increased (p < 0.05) in PM 10 vs. control exposed corneas at 2 weeks. Because we saw biochemical changes in GSH and MDA levels at 2 weeks of exposure, we next examined paraffin-embedded and hematoxylin and eosin-stained corneas at that time period and the data are shown in Figure 2C. PM 10 vs. control exposure showed that the epithelium of the cornea was compacted, nuclei in the superficial corneal layers were pynknotic, and infiltrating cells (containing a brownish particulate, Figure 2C inset) were observed in the PM 10 exposed mice. These cells were easily distinguished due to their distinct cell borders which were visible between corneal epithelial cells which normally are joined by intercellular junctions, including tight junctions. PM 10 vs. control significantly reduced the thickness of the epithelium ( Figure 2D), stroma ( Figure 2E) and the entire cornea ( Figure 2F, p < 0.001 for all). 10 on Innate Immunity mRNA levels of innate immune markers measured after 2 weeks of exposure to control vs. PM 10 are shown in Figure 3A-G. mRNA levels were significantly elevated for chemokine (C-X-C) ligand (CXCL)2 (A, p < 0.001), Toll-like receptor (TLR)4 (B, p < 0.001), TLR2 (C, p < 0.05), interleukin (IL)-6 (D, p < 0.01), and IL-1β (E, p < 0.01) in PM 10 vs. control exposed animals. No significant differences were observed in the levels of tumor necrosis factor (TNF)-α (F) and receptor for advanced glycation end products (RAGE) (G) in PM 10 vs. control animals.

Effects of PM
2.4. Response to PM 10 : Nrf2, GSH Maintenance enzymes and Effects of SKQ1 on Oxidative Stress mRNA levels of GSH maintenance enzymes are shown in Figure 4A-D. Significantly elevated mRNA levels were observed for GSH maintenance enzymes: glutathione peroxidase (GPX) 1 (B, p < 0.01), GPX2 (C, p < 0.01) and glutathione reductase (GR)1 (D, p < 0.001); while Nrf2 (A) levels did not change in PM 10 vs. control exposure at 2 weeks. Figure 4E-G indicates levels of GSH, MDA and Nrf2 protein after 2 weeks of PM 10 vs. control exposure and the effects of the mitochondrial anti-oxidant SKQ1. Figure 4E shows a significant decrease (p < 0.01) in GSH levels after 2 weeks of PM 10 vs. control exposure which are significantly reversed (p < 0.01) by SKQ1 treatment. MDA levels were significantly increased (p < 0.01) after PM 10 vs. control exposure, and treatment with SKQ1 significantly decreased (p < 0.001) MDA levels. Nrf2 protein levels were significantly (<0.01) after PM1-vs. control exposure and SKQ1 restores Nrf2 levels almost to the control level (p < 0.05). The wetted length (red portion) was measured and represented as a bar graph, showed no differences in tear volume. (C) Corneal sensitivity was measured using a Cochet-Bonnet Esthesiometer, showed no significant changes between control and PM10 groups. Data are expressed as mean + SD. (n = 5/group/time).

Effects of PM10 on GSH and MDA Levels and Histopathology
Levels of GSH were significantly lower (p < 0.05) after 2 weeks of exposure to PM10 vs. control (Figure 2A). Levels of MDA ( Figure 2B) were significantly increased (p < 0.05) in PM10 vs. control exposed corneas at 2 weeks. Because we saw biochemical changes in Figure 1. Effects of PM 10 exposure on tear secretion and corneal sensitivity. (A) Phenol red thread images. Red portion of the thread represents tear volume at 0 and 2 weeks after control or PM 10 exposure. (B) The wetted length (red portion) was measured and represented as a bar graph, showed no differences in tear volume. (C) Corneal sensitivity was measured using a Cochet-Bonnet Esthesiometer, showed no significant changes between control and PM 10 groups. Data are expressed as mean + SD. (n = 5/group/time).

Figure 2.
Effects of PM10 exposure: oxidative stress and histology. (A) Levels of GSH were significantly lower after 2 weeks exposure to PM10 vs. control. (B) MDA levels (lipid peroxidation end product) were significantly increased in PM10 vs. control exposed corneas after 2 weeks. (C) Paraffin embedded and hematoxylin and eosin stained corneas after 2 weeks exposure to PM10 or control. Inset shows an infiltrating cell containing a brownish particulate. Scale bar = 20 μm. Thickness of the epithelium (D), stroma (E) and the whole cornea (F) were significantly decreased in PM10 vs. control exposed corneas. Data are expressed as mean + SD. (* p < 0.05, n = 5/group (A,B) *** p < 0.001, n = 3/group (D-F)).

Effects of PM10 on Innate Immunity
mRNA levels of innate immune markers measured after 2 weeks of exposure to control vs. PM10 are shown in Figure 3A-G. mRNA levels were significantly elevated for chemokine (C-X-C) ligand (CXCL)2 (A, p < 0.001), Toll-like receptor (TLR)4 (B, p < 0.001), TLR2 (C, p < 0.05), interleukin (IL)-6 (D, p < 0.01), and IL-1β (E, p < 0.01) in PM10 vs. control exposed animals. No significant differences were observed in the levels of tumor necrosis factor (TNF)-α (F) and receptor for advanced glycation end products (RAGE) (G) in PM10 vs. control animals. Figure 2. Effects of PM 10 exposure: oxidative stress and histology. (A) Levels of GSH were significantly lower after 2 weeks exposure to PM 10 vs. control. (B) MDA levels (lipid peroxidation end product) were significantly increased in PM 10 vs. control exposed corneas after 2 weeks. (C) Paraffin embedded and hematoxylin and eosin stained corneas after 2 weeks exposure to PM 10 or control. Inset shows an infiltrating cell containing a brownish particulate. Scale bar = 20 µm. Thickness of the epithelium (D), stroma (E) and the whole cornea (F) were significantly decreased in PM 10 vs. control exposed corneas. Data are expressed as mean + SD. (* p < 0.05, n = 5/group (A,B) *** p < 0.001, n = 3/group (D-F)).
2.5. Effects of SKQ1 on Cell Viability, Oxidative Stress, Mitochondrial ROS, ATP and Nrf2 Levels after PM 10 Exposure in HCET Phase contrast images of HCET exposed to 100 µg/mL of PM 10 in the presence or absence of SKQ1 are shown ( Figure 5A-C). Figure 5A shows that in the media control group, without PM 10 , cells are spindle-shaped with prominent nuclei. PM 10 -exposed cells appear to be thinned with a few rounded cells, and the nuclei are difficult to see ( Figure 5B). In the presence of SKQ1, PM 10 -exposed cells appear similar to the media control group, with spindle-shaped cells and prominent nuclei ( Figure 5C). HCET exposed to 100µg/mL PM 10 in the absence of SKQ1 showed significantly lower (p < 0.001) cell viability vs. media control ( Figure 5D). Pre-treatment with SKQ1 significantly increased (p < 0.001) the viability of cells exposed to 100 µg/mL PM 10 . Viability was further reduced (p < 0.001) when cells were treated with 200 µg/mL PM 10 vs. media control. However, at 200 µg/mL PM 10 , SKQ1 treatment was unable to positively affect cell viability. MDA levels were significantly increased (p < 0.01) in PM 10 exposed vs. media control ( Figure 5E). SKQ1 significantly reduced (p < 0.05) MDA levels in the PM 10 -exposed cells ( Figure 5E). Figure 5F shows that the levels of mitochondrial ROS were significantly increased (p < 0.001) by PM 10 vs. media control. Pre-treatment with SKQ1 significantly reduced (p < 0.001) levels of mitochondrial ROS ( Figure 5F). PM 10 vs. media control levels significantly lowered (p < 0.001) ATP ( Figure 5G). SKQ1 significantly restored (p < 0.001) ATP levels in PM 10exposed cells ( Figure 5G). The protein levels of Nrf2 measured by Western blot and the integrated density values (IDV) after normalizing Nrf2 to β-tubulin are represented in Figure 5H. Data indicate a significant reduction (p < 0.001) in PM 10 exposed cells vs. media control. Pre-treating HCET with SKQ1 significantly restored (p < 0.001) Nrf2 levels after PM 10 exposure ( Figure 5H).  Figure 3. Effects of PM10 on innate immunity. RT-PCR showed significantly elevated mRNA levels in PM10 vs. control exposed corneas for chemokine CXCL2 (A), TLR4 (B), TLR2 (C), IL-6 (D), IL-1β (E) only but not TNF-α (F) and RAGE (G) after 2 weeks exposure. Data are expressed as mean + SD. (* p < 0.05, ** p < 0.01, *** p < 0.001, n = 5/group).

Response to PM10: Nrf2, GSH Maintenance enzymes and Effects of SKQ1 on Oxidative Stress
mRNA levels of GSH maintenance enzymes are shown in Figure 4A-D. Significantly elevated mRNA levels were observed for GSH maintenance enzymes: glutathione peroxidase (GPX) 1 (B, p < 0.01), GPX2 (C, p < 0.01) and glutathione reductase (GR)1 (D, p < 0.001); while Nrf2 (A) levels did not change in PM10 vs. control exposure at 2 weeks. Figure  4 (E-G) indicates levels of GSH, MDA and Nrf2 protein after 2 weeks of PM10 vs. control exposure and the effects of the mitochondrial anti-oxidant SKQ1. Figure 4E shows a significant decrease (p < 0.01) in GSH levels after 2 weeks of PM10 vs. control exposure which are significantly reversed (p < 0.01) by SKQ1 treatment. MDA levels were significantly increased (p < 0.01) after PM10 vs. control exposure, and treatment with SKQ1 significantly decreased (p < 0.001) MDA levels. Nrf2 protein levels were significantly (<0.01) after PM1vs. control exposure and SKQ1 restores Nrf2 levels almost to the control level (p < 0.05).   Phase contrast images of HCET exposed to 100 μg/mL of PM10 in the presence or

Effects of SKQ1 on Cell Viability and Nrf2 Levels after PM10 Exposure in HCEC
Since we used immortalized corneal epithelial cells (HCET) to study the effects of PM10, we next tested the toxic effects of PM10 on primary human corneal epithelial cells (HCEC). A significant reduction (p < 0.001) in HCEC cell viability was observed upon exposure to 100 μg/mL PM10 vs. media control ( Figure 6A). SKQ1 pre-treatment significantly improved (p < 0.001) the viability after PM10 exposure in HCEC ( Figure 6A). Western blot analysis showed that PM10 vs. media control significantly reduced (p < 0.001) the protein levels of Nrf2 ( Figure 6B). SKQ1 pre-treatment significantly increased (p < 0.01) Nrf2 levels after PM10 exposure vs. PM10 without SKQ1 ( Figure 6B).

Effects of SKQ1 on Cell Viability and Nrf2 Levels after PM 10 Exposure in HCEC
Since we used immortalized corneal epithelial cells (HCET) to study the effects of PM 10 , we next tested the toxic effects of PM 10 on primary human corneal epithelial cells (HCEC). A significant reduction (p < 0.001) in HCEC cell viability was observed upon exposure to 100 µg/mL PM 10 vs. media control ( Figure 6A). SKQ1 pre-treatment significantly improved (p < 0.001) the viability after PM 10 exposure in HCEC ( Figure 6A). Western blot analysis showed that PM 10 vs. media control significantly reduced (p < 0.001) the protein levels of Nrf2 ( Figure 6B). SKQ1 pre-treatment significantly increased (p < 0.01) Nrf2 levels after PM 10 exposure vs. PM 10 without SKQ1 ( Figure 6B).

Discussion
Exposure of the ocular surface to air pollutants can cause significant irritation and discomfort to the eye [24]. While eye diseases do not affect life expectancy, they nonetheless result in a significant reduction in the quality of life [24]. One such condition is dry eye disease, which is triggered by an array of factors, including tear film instability, and is often associated with elevated tear osmotic pressure and ocular inflammation [42]. Epidemiological studies have implicated the role of air pollution in dry eye disease [43] but the exact mechanism by which PM exerts its toxic effects on the cornea is still not completely understood. Different animal models have been developed to study the effects of PM on the eye using topical eye drops [44,45]; however, the topical application does not reflect actual environmental exposure to particulates. To overcome this limitation, we used a whole-body aerosol exposure system to disperse PM10, a well-characterized product purchased from the National Institute of Standards and Technology (NIST, SRM 2787) [46].
A study using PM10 drops at a very high concentration (5 mg/mL, 4X/day) showed reduced tear volume, damaged tear film, impaired ocular surface and reduced goblet cell number in the conjunctiva in male BALB/c mice [47]. However, when we examined the effects of PM10 exposure (500 μg/m 3 in a whole body exposure chamber) on tear secretion, no significant change in tear volume was observed after 2 weeks of exposure. Our results also differ from a recent study in female rats exposed to PM in an exposure chamber (500 μg/m 3 for 2 weeks) that showed a significant reduction in tear volume after 14 days [48]. These differences could be due to the type of particulates used (we used SRM 2787 vs. samples from China), the time of exposure (3 h/d vs. 5 h/d) and different species (mice vs.

Discussion
Exposure of the ocular surface to air pollutants can cause significant irritation and discomfort to the eye [24]. While eye diseases do not affect life expectancy, they nonetheless result in a significant reduction in the quality of life [24]. One such condition is dry eye disease, which is triggered by an array of factors, including tear film instability, and is often associated with elevated tear osmotic pressure and ocular inflammation [42]. Epidemiological studies have implicated the role of air pollution in dry eye disease [43] but the exact mechanism by which PM exerts its toxic effects on the cornea is still not completely understood. Different animal models have been developed to study the effects of PM on the eye using topical eye drops [44,45]; however, the topical application does not reflect actual environmental exposure to particulates. To overcome this limitation, we used a whole-body aerosol exposure system to disperse PM 10 , a well-characterized product purchased from the National Institute of Standards and Technology (NIST, SRM 2787) [46].
A study using PM 10 drops at a very high concentration (5 mg/mL, 4X/day) showed reduced tear volume, damaged tear film, impaired ocular surface and reduced goblet cell number in the conjunctiva in male BALB/c mice [47]. However, when we examined the effects of PM 10 exposure (500 µg/m 3 in a whole body exposure chamber) on tear secretion, no significant change in tear volume was observed after 2 weeks of exposure. Our results also differ from a recent study in female rats exposed to PM in an exposure chamber (500 µg/m 3 for 2 weeks) that showed a significant reduction in tear volume after 14 days [48]. These differences could be due to the type of particulates used (we used SRM 2787 vs. samples from China), the time of exposure (3 h/d vs. 5 h/d) and different species (mice vs. rats). Reduced corneal sensitivity is another characteristic of dry eye disease in patients [49]. However, we did not see any alterations in corneal sensitivity after 2 weeks of PM 10 exposure. Very little is known about the effects of PM 10 on corneal sensitivity in animal models. In our study, histological evaluation of PM 10 -exposed corneas showed pynknotic nuclei and infiltration of inflammatory cells in the epithelium of the cornea. Our data are similar to a previous study that showed increased apoptosis and the infiltration of inflammatory cells in the central cornea of mice that received drops of PM 10 (5 mg/mL) for 2 weeks [47]. Furthermore, we observed that PM 10 -exposed corneas exhibited a reduction in the thickness of the epithelium, stroma and the whole cornea. These data are similar to a previous study that examined the effects of the long-term exposure of PM eye drops (5 mg/mL 4x/d) on the cornea, conjunctiva and retina in rats and found a decreased thickness of the epithelium and whole cornea [40]. To understand how PM 10 exerts its toxicity on the cornea, we then evaluated levels of oxidative stress and inflammation. Our data showed increased pro-inflammatory cytokines and innate immune response markers in the mouse cornea after PM 10 exposure. Similar effects of PM 10 on pro-inflammatory cytokine levels have been reported in the eyes of mice that received PM 10 eye drops for 2 weeks [47]. In addition, we also observed that PM 10 induced oxidative stress (increased MDA levels) by disrupting the anti-oxidant capacity (decreased GSH levels) in the cornea. In this regard, increased lipid peroxidation and reduced GSH levels have been previously reported in the lungs of rats after PM 10 instillation [50]. GSH maintenance and synthesis are regulated by the Nrf2 signaling pathway [51]. Nrf2 regulates the rate of GSH synthesis by controlling the activity of the enzymes required for synthesis namely γ-glutamyl cysteine ligase and glutathione synthetase [51] and also controls GSH maintenance by regulating GPX [52], and GR1 [53]. In our study, PM 10 exposure increased the transcript levels of GPX1, GPX2 and GR1, while Nrf2 levels were unchanged in the mouse cornea after 2 weeks. In contrast, reduced protein levels of Nrf2 were observed which are consistent with data from a mouse model of experimental autoimmune encephalomyelitis (EAE) [54]. Interestingly, low Nrf2 protein levels in EAE were not due to a reduced amount of Nrf2 mRNA, which instead slightly increased in the diseased tissue with time. The discrepancy between mRNA and protein levels for Nrf2 was noted and could be due to alterations in translation and/or other unknown post-translational processes of Nrf2 under pathological conditions such as EAE [54].
Ongoing research is focused on developing eye therapeutics to combat the deleterious effects of air pollution on the eye [55,56]. SKQ1 (Visomitin), a novel mitochondria-targeted anti-oxidant has been used to treat inflammation linked to anesthesia-induced dry eye disease and corneal wounds [40]. In experimental models, SKQ1 administered for 5 days at 50 nmol/kg increased mRNA levels of Nrf2 and antioxidant enzyme genes (SOD1, SOD2, CAT, GPX4) in the cerebral cortex of rat brain under normal and hyperoxic conditions [57].
In our study, we tested the efficacy of SKQ1 as a protective agent against the deleterious effects induced by PM 10 in the mouse cornea. SKQ1 protected the cornea against PM 10 toxicity by significantly reducing oxidative stress (reducing lipid peroxidation) and restoring levels of anti-oxidant GSH and Nrf2.
Since data generated in a mouse system often does not translate to human applicability, we further tested the effects of PM 10 and SKQ1 in vitro on transformed and primary human corneal epithelial cells. PM 10 reduced cell viability in a concentration-dependent manner in transformed corneal epithelial cells. In this respect, PM has been shown to affect cell viability in vitro in a dose-dependent manner in human corneal epithelial cells [58]. We observed that anti-oxidant SKQ1 significantly reversed PM 10 -induced loss in cell viability. Our study showed that SKQ1 was protective when cells were treated with 100 µg/mL, but not 200 µg/mL of PM 10 . Therefore, we selected 100 µg/mL PM 10 as the optimal dose for all further experiments. We further confirmed our findings in primary human corneal epithelial cells and observed that SKQ1 could reverse the toxic effects of PM 10 on cell viability. The protective effects of SKQ1 on the ocular surface have been previously established [59]. SKQ1 can enhance cell proliferation and boost corneal wound healing in corneal limbal epithelial cells at a concentration of 50 nM [59]. Furthermore, the study indicated that at higher concentrations, SKQ1 was cytotoxic [59].
Oxidative stress and inflammation have been implicated as the main mechanisms of PM-mediated toxicity [58]. Components of PM such as poly aromatic hydrocarbons and heavy metals can generate reactive oxygen species (ROS) leading to oxidative stress. Mitochondria are the major generators of ROS, but they are also targets of PM toxicity [60]. In this regard, PM 2.5 can accumulate in the mitochondria, disrupt mitochondrial structure and function, induce mitochondrial ROS and activate the intrinsic apoptotic pathway [32,61]. When we tested PM 10 toxicity in transformed human corneal epithelial cells; we observed increased lipid peroxidation (MDA), increased mitochondrial ROS and reduced ATP production after PM 10 treatment. Impaired mitochondrial function, amplified ROS and increased apoptosis have been previously reported in lung epithelial cells treated with oil fly ash [61] or PM samples collected in Milan [62]. Oxidative stress and inflammation induced by acute or chronic exposure to particulates involve Nrf2 signaling [32,63,64]. At low doses or single acute exposure, PM induces Nrf2 expression [63] but at high doses or multiple chronic exposures, may cause an oxidative burst and thus compromise Nrf2 signaling [32,64]. This can acutely damage mitochondrial redox balance and reduce energy levels [32,65]. We treated both transformed and primary human corneal epithelial cells with a high dose (100µg/mL) of PM 10 and observed a decrease in Nrf2 protein levels. Our data are similar to a study in human alveolar epithelial cells which showed a significant reduction in Nrf2 levels and impairment of GSH after treating cells with 100-500 µg/mL of PM 10 [50]. Additionally, SKQ1 protected cells from oxidative stress, and mitochondrial dysfunction and restored Nrf2 levels after PM 10 exposure. In conclusion, we have shown (diagrammatically in Figure 7) that whole-body exposure to PM 10 triggers oxidative stress and disrupts the Nrf2 pathway in the cornea. SKQ1 treatment reverses these deleterious effects. In vitro human corneal epithelial cell data parallel the in vivo effects. corneal limbal epithelial cells at a concentration of 50 nM [59]. Furthermore, the study indicated that at higher concentrations, SKQ1 was cytotoxic [59].
Oxidative stress and inflammation have been implicated as the main mechanisms o PM-mediated toxicity [58]. Components of PM such as poly aromatic hydrocarbons and heavy metals can generate reactive oxygen species (ROS) leading to oxidative stress. Mi tochondria are the major generators of ROS, but they are also targets of PM toxicity [60] In this regard, PM2.5 can accumulate in the mitochondria, disrupt mitochondrial structure and function, induce mitochondrial ROS and activate the intrinsic apoptotic pathway [32,61]. When we tested PM10 toxicity in transformed human corneal epithelial cells; we observed increased lipid peroxidation (MDA), increased mitochondrial ROS and reduced ATP production after PM10 treatment. Impaired mitochondrial function, amplified ROS and increased apoptosis have been previously reported in lung epithelial cells treated with oil fly ash [61] or PM samples collected in Milan [62]. Oxidative stress and inflam mation induced by acute or chronic exposure to particulates involve Nrf2 signaling [32,63,64]. At low doses or single acute exposure, PM induces Nrf2 expression [63] but a high doses or multiple chronic exposures, may cause an oxidative burst and thus compro mise Nrf2 signaling [32,64]. This can acutely damage mitochondrial redox balance and reduce energy levels [32,65]. We treated both transformed and primary human cornea epithelial cells with a high dose (100μg/mL) of PM10 and observed a decrease in Nrf2 pro tein levels. Our data are similar to a study in human alveolar epithelial cells which showed a significant reduction in Nrf2 levels and impairment of GSH after treating cells with 100-500 μg/mL of PM10 [50]. Additionally, SKQ1 protected cells from oxidative stress, and mi tochondrial dysfunction and restored Nrf2 levels after PM10 exposure. In conclusion, we have shown (diagrammatically in Figure 7) that whole-body exposure to PM10 triggers oxidative stress and disrupts the Nrf2 pathway in the cornea. SKQ1 treatment reverses these deleterious effects. In vitro human corneal epithelial cell data parallel the in vivo effects.

Mice
Eight-week-old C57BL/6 female mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA) and housed in accordance with the National Institutes of Health guidelines. They were humanely treated and in compliance with both the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research) and the Institutional Animal Care and Use Committee of Wayne State University (IACUC 21-09-4042).

Whole Body Exposure to PM 10
Particulate matter, a major air pollutant is a general term for small atmospheric solid and liquid particles that vary in size (2.5-10 µm), composition and origin [66]. All experiments in this study were performed with PM 10 purchased from the National Institute of Standards and Technology, (NIST) (Standard Reference Material (SRM) 2787). A wholebody exposure chamber (CH Technologies, Westwood, NJ, USA) was used for all the in vivo experiments performed in this study (Figure 8). Exposures were carried out in a stainless steel, whole-body inhalation chamber; one group was exposed to PM 10 whereas the other received only humidified compressed air (control). The apparatus is equipped with a Vilnius aerosol generator (VAG), a dry powder dispenser that offers high stability of aerosol output. Mice were exposed to control or an acute, high dose of PM 10 (500 µg/m 3 ) for 2 weeks for 3 h/day/5 days/week and rested on the weekends. For the in vivo study, the dosage was based on mean PM 10 concentrations measured in the winter, ranging as high as 494 µg/m 3 in five Chinese cities [67]. For in vitro studies, PM 10 was used at a concentration of 100 µg/mL as previously published [68,69].

Mice
Eight-week-old C57BL/6 female mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA) and housed in accordance with the National Institutes of Health guidelines. They were humanely treated and in compliance with both the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research) and the Institutional Animal Care and Use Committee of Wayne State University (IACUC 21-09-4042).

Whole Body Exposure to PM10
Particulate matter, a major air pollutant is a general term for small atmospheric solid and liquid particles that vary in size (2.5-10 μm), composition and origin [66]. All experiments in this study were performed with PM10 purchased from the National Institute of Standards and Technology, (NIST) (Standard Reference Material (SRM) 2787). A wholebody exposure chamber (CH Technologies, Westwood, NJ, USA) was used for all the in vivo experiments performed in this study (Figure 8). Exposures were carried out in a stainless steel, whole-body inhalation chamber; one group was exposed to PM10 whereas the other received only humidified compressed air (control). The apparatus is equipped with a Vilnius aerosol generator (VAG), a dry powder dispenser that offers high stability of aerosol output. Mice were exposed to control or an acute, high dose of PM10 (500 μg/m 3 ) for 2 weeks for 3 h/day/5 days/week and rested on the weekends. For the in vivo study, the dosage was based on mean PM10 concentrations measured in the winter, ranging as high as 494 μg/m 3 in five Chinese cities [67]. For in vitro studies, PM10 was used at a concentration of 100 μg/mL as previously published [68,69].

SKQ1 Treatment
SKQ1 (BOC Sciences, Shirley, NY, USA) was used to treat PM10-exposed mice using a published dose of 7.5 μM [40]. Eyes were treated topically with 5 μL SKQ1 or PBS (control), three times a day before the first chamber exposure and then once each day before exposure for 2 weeks. For in vitro studies, SKQ1 was used at a concentration of 50 nM as previously described [59].

SKQ1 Treatment
SKQ1 (BOC Sciences, Shirley, NY, USA) was used to treat PM 10 -exposed mice using a published dose of 7.5 µM [40]. Eyes were treated topically with 5 µL SKQ1 or PBS (control), three times a day before the first chamber exposure and then once each day before exposure for 2 weeks. For in vitro studies, SKQ1 was used at a concentration of 50 nM as previously described [59].

Endotoxin and Beta-D-Glucan
We are using Standard Reference Material (SRM) 2787 National Institute of Standards and Technology, (NIST) which is one of the most characterized particulate materials for a wide range of organic and inorganic constituents [46].
The mass fractions of the constituents represent those in a contemporary urban environment and complement the mass fractions found in existing SRM. To determine levels of endotoxin, we also tested SRM 2787 using the Pyrochrome Kit for detection and quantification of Gram-negative bacterial endotoxin (Associates of Cape Cod Inc. Falmouth, MA, USA) and endotoxin (E. coli O113:H10) to create a standard curve (ACCI). PM 10 was tested at concentrations of 5 µg/mL, 1 µg/mL, 0.5 µg/mL (equivalent to 500 µg/m 3 , used for whole-body exposure), 0.25 µg/mL, and 0.125 µg/mL. All concentrations tested were below the limit of detection (0.005 EU/mL). We also tested for fungal contaminant (1,3)-Beta-D-Glucan using the Glucatell (1,3)-Beta-D-Glucan Kit (Associates of Cape Cod Inc.). PM 10 was tested at the same concentrations used for testing endotoxin. All concentrations were below the limit of detection (0.625 pg/mL β-glucan standard, not shown). These data assure PM 10 from NIST is not contaminated by either bacterial or fungal components which would complicate data analysis.

Tear Secretion
Tear levels were measured in the right eye of the control and PM 10 exposed mice (n = 5/group/treatment) using Zone-Quick phenol red treated threads (Tianjin Jingming New Technological Development Co., Ltd., Tianjin, China) as described before [70]. Measurements were made before chamber exposure (week 0) to acquire a baseline reading and then at 2 weeks of exposure. Briefly, mice were lightly anesthetized and held while tear levels were measured by placing the tip of a phenol red impregnated cotton thread with a blunt tip on the bulbar conjunctiva (lateral canthus for 30 s). Appropriate care was taken to avoid touch stimulation of eyelashes and whiskers. The wetted length (in millimeters), indicated by a color change from yellow to red, was photographed and measured. Data are shown as mean wetted length + SD.

Corneal Sensation
Corneal sensitivity was measured for control and PM 10 exposed mice (n = 5/group/treatment) using a Cochet-Bonnet esthesiometer (Luneau SAS #10129; Prunay-le-Gillon, France) as described before [70]. Measurements were made before chamber exposure (week 0) to acquire a baseline reading and at 2 weeks after exposure. Measurements were made from a filament length of 60 mm and gradually decreased in 5 mm steps to a length of 5 mm. For each filament length, five repeat measurements were conducted. If three blinks were evoked in response to five consecutive touches, a positive response was recorded. The longest filament length which caused a positive response was recorded as the corneal sensitivity threshold. Data are shown as mean filament length + SD.

Reduced Glutathione (GSH) Assay
Total GSH levels were analyzed by a glutathione assay kit (Cayman Chemical, Ann Arbor, MI, USA) per the manufacturer's protocol and as previously described [71]. Briefly, individual mouse corneas (n = 5/group/treatment) from control vs. PM 10 exposed groups ± SKQ1 were harvested in 500 µL of ice-cold 50 mM MES (2-(N-morpholino) ethanesulphonic acid) containing 1 mM EDTA, homogenized and centrifuged at 10,000× g at 4 • C for 10 min and the supernatant was collected. Total GSH levels were determined using Ellman's reagent (DTNB, 5,5 -dithio-bis-2-nitrobenzoic acid), which reacts with the sulfhydryl group of GSH resulting in a yellow-colored 5-thio-2-nitrobenzoic acid (TNB) product. Measurement of the absorbance of TNB at 405 nm provides an estimate of GSH in the sample. GSH levels were calculated using a standard curve and then normalized to the total protein in each sample. A Bradford assay was used to determine protein concentration in each sample. Protein levels were calculated using a standard curve with bovine serum albumin and Bio Rad protein assay reagent (Bio Rad, Hercules, CA, USA) per the manufacturer's protocol. Final GSH levels were expressed as mean + SD.

ELISA
ELISA kits were used to measure protein levels of Nrf2 (Novus Biologicals, Centennial, CO, USA) and lipid peroxidation end product MDA (Cell Biolabs, San Diego, CA, USA). Briefly, individual mouse corneas (n = 5/group/treatment) from the control vs. PM 10 exposed groups ± SKQ1 were harvested in 500 µL of PBS containing 0.1% Tween 20 and protease inhibitors. All assays were run per the manufacturer's protocol and data were normalized to total protein (described above) and expressed as mean + SD.

H&E Staining of Exposed Corneas
Whole eyes (n = 3) were harvested from mice after 2 weeks of exposure to control or 500 µg/m 3 PM 10 . Eyes were fixed in alcoholic z-fix and sent to Excalibur Pathology, Inc (Norman, OK) where they were embedded in paraffin, sectioned and stained with H&E. Photographs of representative areas of the epithelium were taken using a Leica DM4000B light microscope at 20X. The thickness of the whole cornea, epithelium and stroma were analyzed from three mice in each group (72 measurements/group/treatment). Data are represented as thickness (in mm) and expressed as mean + SD.

RT-PCR
Total RNA was isolated from control and PM 10 -exposed mouse corneas (n = 5/group/treatment) (RNA STAT-60; Tel-Test, Friendswood, TX, USA) per the manufacturer's instructions as reported before [72]. Briefly, 1 µg of each RNA sample was reverse transcribed using Moloney-murine leukemia virus (M-MLV) reverse transcriptase (Invitrogen, Carlsbad, CA, USA) to produce a cDNA template and diluted 1:20 using DEPC-treated water. A 2 µL aliquot of diluted cDNA was used for the RT-PCR reaction. SYBR green/fluorescein PCR master mix (Bio-Rad Laboratories, Richmond, CA, USA) and primer concentrations of 10 µM were used in a total 10 µL volume. After a pre-programmed hot start cycle (3 min at 95 • C), PCR amplification was repeated for 45 cycles with parameters: 15 s at 95 • C and 60 s at 60 • C. Levels of chemokine CXCL2, TLR4, TLR2, IL-6, IL-1β, TNF-α, RAGE, Nrf2, GPX1, GPX2, and GR1 were tested by real-time RT-PCR (CFX Connect real-time PCR detection system; Bio-Rad Laboratories). The fold differences in gene expression were calculated relative to housekeeping gene β-actin mRNA and expressed as the relative mRNA concentration + SD. Primer pair sequences used are shown in Table 1.

Tissue Culture
HCET cells (HCE-2 [50.B1], ATCC, Gaithersburg, MA, USA) were cultured in Keratinocyteserum free medium (Gibco, Grand Island, NY, USA) with 5 ng/mL human recombinant EGF, 0.05 mg/mL bovine pituitary extract, 0.005 mg/mL insulin, and 500 ng/mL hydrocortisone as reported before [73]. HCEC (primary corneal epithelial cells, ATCC) were cultured in Keratinocyte-serum free medium (Gibco) with 5 ng/mL human recombinant EGF and 0.05 mg/mL bovine pituitary extract as per the manufacturer's protocol. To evaluate the effects of SKQ1, a subset of cells was incubated with 50 nM SKQ1 as previously described [59], 1 h prior to PM 10 exposure. Phase contrast microscopy was used to photograph cell preparations using a Leica EL 6000 microscope (Deerfield, IL, USA).

Cell Viability Assay
An MTT 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (ThermoFisher Scientific, Grand Island, NY, USA) assay was used to test the effects of PM 10 on HCET viability in the presence and absence of SKQ1, per the manufacturer's protocol and as reported before [73]. Briefly, 15,000 HCET cells were seeded in a 96-well plate, treated with increasing concentrations of PM 10 (0, 100 and 200 µg/mL) ± 50 nM SKQ1 and incubated for 24 h. At the end of the treatments, 5 mg/mL MTT reagent was added to each well and incubated at 37 • C for 4 h and the media was removed. Dimethyl sulfoxide (DMSO) was added (50 µL/well) and optical density was read at 540 nm using a SpectraMax M5 microplate reader (Molecular Devices, Sunnyvale, CA, USA). The cell viability was tested similarly for HCEC with 0 and 100 µg/mL PM 10 ± 50 nM SKQ1 after 24 h incubation. Data are shown as % cell viability + SD. Table 1. Nucleotide sequence of the specific primers used for PCR amplification (Mouse).

Gene
Nucleotide

Mitochondrial ROS Assay
Mitochondrial ROS was analyzed by fluorescence with MitoSOX assay (ThermoFisher Scientific) according to the manufacturer's protocol and as previously described [63]. Briefly, 15,000 HCET cells were plated onto 96 well plates, and treated with 100 µg/mL PM 10 ± 50 nM SKQ1 for 6 h. Cells were washed with PBS and incubated with 10 µM MitoSOX at 37 • C for 10 min. Cells were then washed twice with PBS and fluorescence was measured at 510 nm (excitation) and 590 nm (emission) using SpectraMax M5 spectrophotometer (Molecular Devices). The level of ROS is proportional to fluorescence intensity. Data were normalized to control and are represented as % relative fluorescence + SD.

Mitochondrial ToxGlo Assay
To determine whether PM 10 caused a reduction in ATP levels, a mitochondrial ToxGlo assay (Promega, Madison, WI, USA) was performed per the manufacturer's protocol and as previously described [74]. Briefly, 15,000 HCET were plated onto 96 well plates, incubated overnight and media removed. Cells were then incubated galactose supplemented RPMI 1640 media and treated with 100 µg/mL PM 10 ± 50 nM SKQ1 for 3 h. ATP detection reagent was added to each well, mixed and incubated for 5 min and luminescence was measured. The levels of ATP are represented as relative luminescence. Data are shown as mean + SD.

Statistical Analysis
For in vivo data analysis, a one-way ANOVA followed by Bonferroni's multiple comparison test (GraphPad Prism) was used to test the significance of tear production, corneal sensitivity, GSH, and ELISA (Nrf2, MDA). A Student's t-test (GraphPad Prism) was used to determine the significance of the thickness of the whole cornea, epithelium, stroma, RT-PCR, GSH assay and ELISA (MDA) in vivo. For analyzing in vitro data, a one-way ANOVA followed by Bonferroni's multiple comparison test (GraphPad Prism) was used to test the significance for MTT, ROS, ATP assay, ELISA (MDA) and Western blot. Data were considered significant at p < 0.05. All experiments were repeated at least once to ensure reproducibility and data are shown as mean + SD.